Sorting process of nanoparticles and applications of same

ABSTRACT

A sensing platform includes a substrate, an array of wells formed in the substrate, and sorted nanoparticles filling in the array of wells such that each well contains a single sorted nanoparticle, wherein the sorted nanoparticles are in a cluster state.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a divisional patent application of, and claimsbenefit of U.S. patent application Ser. No. 13/461,521, filed May 1,2012, entitled “SORTING PROCESS OF NANOPARTICLES AND APPLICATIONS OFSAME”, by Timothy P. Tyler et al., now U.S. Pat. No. 9,802,818, whichitself claims priority to and the benefit of, pursuant to 35 U.S.C. §119(e), U.S. Provisional Patent Application Ser. No. 61/481,994, filedMay 3, 2011, entitled “IMPROVED MONODISPERSITY OF CORE/SHELLNANOPARTICLES VIA CERTRIFUGAL PROCESSING”, by Timothy P. Tyler et al.,which are incorporated herein in their entireties by reference.

STATEMENT AS TO RIGHTS UNDER FEDERALLY-SPONSORED RESEARCH

This invention was made with government support under FA9550-08-1-0221awarded by the Air Force Office of Scientific Research, DE-SC0001059awarded by the Department of Energy, and CHE0911145, DMR0520513, andDMR1006391 awarded by the National Science Foundation. The governmenthas certain rights in the invention.

Some references, which may include patents, patent applications andvarious publications, are cited and discussed in the description of thisinvention. The citation and/or discussion of such references is providedmerely to clarify the description of the present invention and is not anadmission that any such reference is “prior art” to the inventiondescribed herein. All references cited and discussed in thisspecification are incorporated herein by reference in their entiretiesand to the same extent as if each reference was individuallyincorporated by reference. In terms of notation, hereinafter, “[n]”represents the nth reference cited in the reference list. For example,[27] represents the 27-th reference cited in the reference list, namely,Arnold, M. S.; Green, A. A.; Hulvat, J. F.; Stupp, S. I.; Hersam, M. C.Sorting Carbon Nanotubes by Electronic Structure Using DensityDifferentiation. Nature Nanotech. 2006, 1, 60-65.

FIELD OF THE INVENTION

The present invention relates generally to nanoparticles, and moreparticularly to centrifugal sorting processes of core/shellnanoparticles and applications of the same.

BACKGROUND OF THE INVENTION

The intense electromagnetic field arising at the surface of metallicnanostructures from the excitation of the localized surface plasmonresonance (LSPR) allows for the enhancement of the Raman intensity ofadsorbed molecules by a factor up to 4×10⁸ or greater [1]. Thestructures supporting this plasmonic phenomenon, known assurface-enhanced Raman scattering (SERS), are diverse. The mostsensitive examples, with an enhancement factor large enough to observespectra at the single molecule level [2-5], are aggregated metallicnanoparticles. Correlative structure-activity studies have indeed shownthat the presence of a nanometer-sized junction [6, 7] or crevice [1, 8]creates the electromagnetic ‘hot-spot’ (i.e., ‘nanoantenna’) required toobserve single molecule SERS. Recent investigations of the hot-spots atthe junction of silver cubes [9] and gold pyramidal shells [10] or atthe interface between a gold nanostar and a gold surface [11] havehighlighted how the control over the structure of this nanometer-scaleregion is crucial for achieving high enhancement factors. While theearly fundamental studies of single molecule SERS have been performed oninhomogeneous samples, the integration of plasmonic nanoantennas intoreliable technological applications, such as high sensitivity biologicaland chemical sensors, requires improved structural reproducibility.

Homogeneous nanostructure populations can be realized via preciselycontrolled fabrication or post-synthetic sorting techniques. Althoughmuch effort has been devoted to the controlled synthesis ofnanoparticles, structural polydispersity remains an issue [11-14].Consequently, post-fabrication separation methods have become importantfor characterizing or refining populations of nanoparticles based ontheir size, shape, and aggregation state [15] For example,electrophoretic methods [16], most notably gel electrophoresis [17],have been used to separate metal nanoparticles by both size and shape.Size-exclusion chromatography has also been demonstrated for separatinggold nanoparticles by shape [18] and as a tool for characterizingsynthesized nanoparticles [19]. In addition, sedimentation coefficientdifferences between nanoparticles of varying size and shape have beenexploited for sorting by centrifugation [20] and sedimentationfield-flow fractionation [21]. In particular, a recent study onpolymer-coated nanoparticle clusters employed centrifugation andfiltration to remove single-core nanoparticles and large aggregatesrespectively, ultimately yielding samples of primarily multi-corenanoparticle clusters with enhanced SERS signals [22]. Finally, densitygradient centrifugation has proven to be particularly successful forobtaining refined populations of nanoparticles, leading to narrowdiameter and shape distributions [23, 24] or a specific aggregationstate for nanoparticle clusters [25].

For plasmonic applications, the removal of single nanoparticles fromaggregates is particularly desirable given that only nanoparticleaggregates (i.e., two or more metallic nanoparticles) have thus far beenshown to provide sufficient enhancement for single molecule and singleparticle SERS [6]. Improved monodispersity within nanoantenna samplesallows for increased ensemble SERS signals and removal of inactivespecies for potential sensing applications. Efforts toward improvedmonodispersity through controlled synthesis of nanoparticles have beenunable to fully address this issue. Previous centrifugal sorting methodsfor nanoparticles have been limited to slower-sedimenting small diameternanoparticles and have required chemical functionalization orsurfactants to keep the nanoparticles dispersed in solution.

Therefore, a heretofore unaddressed need exists in the art to addressthe aforementioned deficiencies and inadequacies.

SUMMARY OF THE INVENTION

The present invention discloses, among other things, an aqueoussurfactant-free centrifugal sorting method for plasmonic nanoantennasincluding silica-coated gold nanoparticle clusters that yieldspopulations of predominantly one aggregation state and an enhancedensemble SERS response. By using a high-viscosity medium, such asiodixanol, one is able to sort the relatively massive gold/silicananoantennas by sedimentation coefficient via transient density gradientcentrifugation, thus producing samples with a preponderance of aselected aggregation state. Furthermore, the silica shell allows thenanoparticles to be dispersed in water without further functionalizationor surfactants, and also protects the SERS reporter molecules at thegold/silica interface. SERS ensemble measurements confirm an increasedsignal from fractions with a diminished monomer population that, whencombined with improved control over nanoparticle cluster size, providesa route to improved reliability and reproducibility in plasmonicapplications such as SERS-based sensors. This sorting approach can beapplied to a wide variety of core/shell nanoparticle structures ingeneral to achieve more monodisperse samples for relatively largenanoparticles.

In one aspect of the invention, a method for sorting nanoparticlescomprises preparing an aqueous iodixanol density gradient medium filledin at least one centrifugal tube; dispersing nanoparticles into anaqueous solution to form a suspension of the nanoparticles, wherein eachnanoparticle comprises one or more gold cores and a silica shellencapsulating the one or more gold cores; layering the suspension of thenanoparticles on the top of the aqueous iodixanol density gradientmedium in the at least one centrifugal tube; centrifugating the layeredsuspension of the nanoparticles at a predetermined speed for apredetermined period of time to form a gradient of fractions of thenanoparticles in the at least one centrifugal tube, wherein eachfraction comprises nanoparticles in a respective one of aggregationstates of the nanoparticles; wherein each aggregation state is a monomerstate or a cluster state that ranges from dimers to dodecamers; andcollecting each fraction of the nanoparticles from the at least onecentrifugal tube.

Each of the one or more gold cores has a size/diameter in a range ofabout 10 nm to about 500 nm. The silica shell has a thickness in a rangeof about 10 nm to about 150 nm.

In one embodiment, each nanoparticle further comprises SERS reportermolecules hosted at the interface of the gold core and the silica shell,where the SERS reporter molecules comprises (1,2-bis(4-pyridyl)ethylene(BPE)).

In one embodiment, the aqueous iodixanol density gradient mediumcomprises about 30%-60% weight per volume iodixanol.

In another aspect of the invention, a method for sorting nanoparticlesincludes preparing a high-viscosity density gradient medium filled in acontainer; dispersing nanoparticles into an aqueous solution to form asuspension of the nanoparticles, wherein each nanoparticle comprises oneor more cores and a shell encapsulating the one or more cores; layeringthe suspension of the nanoparticles on the top of the high-viscositydensity gradient medium in the container; and centrifugating the layeredsuspension of the nanoparticles on the top of the high-viscosity densitygradient medium in the container at a predetermined speed for apredetermined period of time to form a gradient of fractions of thenanoparticles along the container, wherein each fraction comprisesnanoparticles in a respective one of aggregation states of thenanoparticles.

Additionally, the method further includes collecting each fraction ofthe nanoparticles from the container.

In one embodiment, each of the one or more cores is formed of a noblemetal, such as gold. In one embodiment, the shell is formed of amaterial such that the nanoparticles are dispersed in the aqueoussolution without need for functionalization or surfactants. For example,the shell is formed of silicon.

In one embodiment, each nanoparticle further comprises SERS reportermolecules hosted at the interface of the core and the shell, where theSERS reporter molecules comprises (1,2-bis(4-pyridyl)ethylene (BPE)).

In one embodiment, each of the one or more cores has a size/diameter ina range of about 10 nm to about 500 nm, and wherein the shell has athickness in a range of about 10 nm to about 150 nm. The nanoparticlesare biocompatible.

In one embodiment, each of the aggregation states is corresponding to amonomer state or a cluster state that ranges from dimers to dodecamers.

In one embodiment, the high-viscosity density gradient medium comprisesan aqueous iodixanol density gradient medium, wherein the aqueousiodixanol density gradient medium comprises about 30%-60% weight pervolume iodixanol.

In one embodiment, the container comprises one or more centrifugaltubes.

In yet another aspect of the invention, a sensing platform comprises asubstrate; an array of wells formed in the substrate; and sortednanoparticles filling in the array of wells such that each well containsa single sorted nanoparticle, wherein the sorted nanoparticles are in acluster state. The cluster state corresponds to one of a dimer state toa dodecamer state. The sorted nanoparticles are biocompatible.

In one embodiment, each sorted nanoparticle comprises two or more coresand a shell encapsulating the two or more cores. Each of the two or morecores is formed of a noble metal, such as gold. The shell is formed of amaterial such that the nanoparticles are dispersed in the aqueoussolution without need for functionalization or surfactants. In oneembodiment, the shell is formed of silicon.

In one embodiment, each sorted nanoparticle further comprises SERSreporter molecules hosted at the interface of the two or more cores andthe shell, wherein the SERS reporter molecules comprises(1,2-bis(4-pyridyl)ethylene (BPE)).

In a further aspect of the invention, a microfluidic device comprisesthe sensing platform disclosed above.

These and other aspects of the present invention will become apparentfrom the following description of the preferred embodiment taken inconjunction with the following drawings, although variations andmodifications therein may be affected without departing from the spiritand scope of the novel concepts of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate one or more embodiments of theinvention and together with the written description, serve to explainthe principles of the invention. Wherever possible, the same referencenumbers are used throughout the drawings to refer to the same or likeelements of an embodiment.

FIG. 1 shows characterization of the as-synthesized gold core/silicashell nanoparticle sample: (a) a representative TEM image showing thestructure and variation in the number of gold cores within each silicashell, (b) a histogram of the populations as a function of the number ofcores, and (c) an extinction spectrum of the as-synthesized sampledispersed in water.

FIG. 2 shows (a) a photograph of a centrifuge tube after centrifugationof the sample in a surfactant-free aqueous iodixanol linear densitygradient, (b) extinction spectra of three selected fractions, (i), (ii),and (iii), whose positions in the centrifugation tube are spatiallyindicated, (c) corresponding TEM images of the selected fractions, and(d) corresponding population histograms of the selected fractions.

FIG. 3 shows centrifuge tube images (a) and (d) of separations runningat 10 minutes and 20 minutes, respectively. Corresponding fractions arespatially indicated, each accompanied by a sample TEM image (b)/(e) andhistogram (c)/(f) of aggregation states. A modest improvement in sortingis observed, although the concentration of dimers remains under 75%.

FIG. 4 shows aggregates assembled in a line, or in a random non-linearorientation. For this analysis, all aggregates were approximated asprolate ellipsoids with an appropriate aspect ratio derived from the TEMdata.

FIG. 5 shows a histogram of sedimentation coefficients for varyingaggregation states, calculated by applying a Perrin ellipsoid model toindividually measured nanoclusters. This model predicts high purityseparation of monomers, followed by overlapping domains of dimers,trimers, tetramers, and higher order clusters, in agreement with theexperimental data.

FIG. 6 shows measured SER spectra of 1,2-bis(4-pyridyl)ethylene (BPE),from (a) fraction (i), (b) the initial solution, (c) fraction (ii)enriched in dimers, and (d) fraction (iii) enriched in tetramers whenilluminated at a 632.8 nm excitation wavelength and power of 6.0 mW.

FIG. 7 shows schematic of sorted nanoantennas filling a welled sensingsubstrate according to one embodiment of the invention.

FIG. 8 shows an SEM image of a welled substrate containing unsortednanoantennas, where the vast majority is monomers, i.e. inactive.

FIG. 9 shows an SEM image of a welled substrate containing centrifugallysorted nanoantennas according to one embodiment of the invention, wherethe population of inactive species (monomers) is minimal.

FIG. 10 shows an optical microscope image of a microfluidic deviceincorporating a nanoantenna-in-wells substrate (inset) according to oneembodiment of the invention. Raman spectra have successfully beenmeasured, demonstrating that this detection system is compatible withthe microfluidic architecture.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is more particularly described in the followingexamples that are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art. Various embodiments of the invention are now described indetail. Referring to the drawings, like numbers indicate like componentsthroughout the views. Additionally, titles or subtitles may be used inthe specification for the convenience of a reader, which shall have noinfluence on the scope of the present invention.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used. Certain terms that are used todescribe the invention are discussed below, or elsewhere in thespecification, to provide additional guidance to the practitionerregarding the description of the invention. For convenience, certainterms may be highlighted, for example using italics and/or quotationmarks. The use of highlighting has no influence on the scope and meaningof a term; the scope and meaning of a term is the same, in the samecontext, whether or not it is highlighted. It will be appreciated thatsame thing can be said in more than one way. Consequently, alternativelanguage and synonyms may be used for any one or more of the termsdiscussed herein, nor is any special significance to be placed uponwhether or not a term is elaborated or discussed herein. Synonyms forcertain terms are provided. A recital of one or more synonyms does notexclude the use of other synonyms. The use of examples anywhere in thisspecification including examples of any terms discussed herein isillustrative only, and in no way limits the scope and meaning of theinvention or of any exemplified term. Likewise, the invention is notlimited to various embodiments given in this specification.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the relevant art and thepresent disclosure, and will not be interpreted in an idealized oroverly formal sense unless expressly so defined herein.

As used in the description herein and throughout the claims that follow,the meaning of “a”, “an”, and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein and throughout the claims that follow, the meaning of “in”includes “in” and “on” unless the context clearly dictates otherwise.

It will be understood that when an element is referred to as being “on”another element, it can be directly on the other element or interveningelements may be present therebetween. In contrast, when an element isreferred to as being “directly on” another element, there are nointervening elements present. As used herein, the term “and/or” includesany and all combinations of one or more of the associated listed items.

It will be understood that, although the terms first, second, third etc.may be used herein to describe various elements, components, regions,layers and/or sections, these elements, components, regions, layersand/or sections should not be limited by these terms. These terms areonly used to distinguish one element, component, region, layer orsection from another element, component, region, layer or section. Thus,a first element, component, region, layer or section discussed belowcould be termed a second element, component, region, layer or sectionwithout departing from the teachings of the present invention.

As used herein, “around”, “about” or “approximately” shall generallymean within 20 percent, preferably within 10 percent, and morepreferably within 5 percent of a given value or range. Numericalquantities given herein are approximate, meaning that the term “around”,“about” or “approximately” can be inferred if not expressly stated.

As used herein, if any, the term “transmission electron microscopy” orits abbreviation “TEM” refers to a microscopy technique whereby a beamof electrons is transmitted through an ultra thin specimen, interactingwith the specimen as it passes through. An image is formed from theinteraction of the electrons transmitted through the specimen; the imageis magnified and focused onto an imaging device, such as a fluorescentscreen, on a layer of photographic film, or to be detected by a sensorsuch as a CCD camera.

As used herein, the term “surface enhanced Raman spectroscopy” or itsabbreviation “SERS” refers to a surface-sensitive technique thatenhances Raman scattering by molecules adsorbed on rough metal surfaces.The enhancement factor can be as much as 10¹⁰ to 10¹¹, which means thetechnique may detect single molecules.

As used herein, if any, the term “scanning electron microscope” or itsabbreviation “SEM” refers to a type of electron microscope that imagesthe sample surface by scanning it with a high-energy beam of electronsin a raster scan pattern. The electrons interact with the atoms thatmake up the sample producing signals that contain information about thesample's surface topography, composition and other properties such aselectrical conductivity.

As used herein, “nanoscopic-scale”, “nanoscopic”, “nanometer-scale”,“nanoscale”, “nanocomposites”, “nanoparticles”, the “nano-” prefix, andthe like generally refers to elements or objects of intermediate sizebetween molecular and microscopic (micrometer-sized) structures, havingwidths or diameters of less than about 1 μm, preferably less than about100 nm in some cases. In all embodiments, specified widths can besmallest width (i.e. a width as specified where, at that location, thearticle can have a larger width in a different dimension), or largestwidth (i.e. where, at that location, the object's width is no wider thanas specified, but can have a length that is greater).

As used herein, the terms “comprising”, “including”, “carrying”,“having”, “containing”, “involving”, and the like are to be understoodto be open-ended, i.e., to mean including but not limited to.

Overview of the Invention

Noble metal nanoparticle clusters underlie a variety of plasmonicdevices and measurements including surface-enhanced Raman spectroscopy(SERS). Due to the strong dependence of plasmonic properties onnanoparticle cluster aggregation states, the elimination ofnon-SERS-active structures and the refinement of the nanoparticlecluster population are critical to realizing uniform and reproduciblestructures for plasmonic nanoantenna applications such as SERS-basedsensors. SERS nanoantennas including aggregated spherical gold coresencapsulated in a protective silica shell with Raman reporter moleculesadsorbed at the gold/silica interface have been shown to be ideal SERSsubstrates [1, 26], offering robustness and stability. The silica shellhas the added benefit that it directly enables dispersion in aqueoussolutions, thereby eliminating the need for additional chemicalfunctionalization. While equilibrium isopycnic density gradientcentrifugation techniques that are common for carbon-based nanomaterials[27-32] are incompatible with high-density structures such asnanoparticle clusters, sorting can, in principle, be accomplished bysedimentation coefficient in the transient centrifugal regime. However,in this study, the relatively large size of the gold cores (about 100 nmin diameter) combined with the silica shell (about 60 nm thick) makessorting by transient motion challenging since these high-mass structureswill sediment significantly faster than smaller nanoparticles. Accordingto the invention, the issue is overcome by using the high-viscositydensity gradient medium iodixanol, which slows the sedimentation of thegold/silica nanoparticle clusters to the point where structurallydistinct fractions can be collected following centrifugation.

In one aspect, the invention relates to a centrifugal sorting techniquefor gold core/silica shell nanoparticles that host SERS reportermolecules at the gold/silica interface. In one embodiment, relativelymassive nanoparticle clusters are sorted by sedimentation coefficientvia centrifugation in a high-viscosity density gradient medium, such asan aqueous iodixanol density gradient medium, which yields solutionsthat contain a preponderance of one aggregation state and a diminishedmonomer population as determined by transmission electron microscopy(TEM), extinction spectroscopy, and SERS. A quantitative analysis of thenanoparticle sedimentation coefficients is presented, thus allowing thisapproach to be predictably generalized to other nanoparticle systems.According to the invention, the collected fractions are found to possessa preponderance of one aggregation state and a diminished monomerpopulation, thus yielding ideal SERS nanoantennas that can be directlyemployed in a variety of plasmonic applications. In addition, thenanoparticle sedimentation coefficients are quantitatively analyzed,which facilitate the applications of this approach to other nanoparticlesystems.

Accordingly, the sorting method includes preparing a high-viscositydensity gradient medium filled in a container including, for example,one or more centrifugal tubes. In one embodiment, the high-viscositydensity gradient medium is formed of about 30%-60% weight per volumeiodixanol. Other composites can also be utilized to practice theinvention.

The sorting method also includes dispersing nanoparticles to be sortedinto an aqueous solution to form a suspension of the nanoparticles. Thenanoparticles exist in a variety of aggregation states, whichcorresponds to monomers or clusters that ranges from dimers tododecamers. Accordingly, each nanoparticle comprises one or more coresand a shell encapsulating the one or more cores. In one embodiment, eachof the one or more cores is formed of a noble metal, such as gold. Theshell is formed of a material such that the nanoparticles are dispersedin the aqueous solution without need for functionalization orsurfactants. For example, the shell is formed of silicon. Each of theone or more cores has a size/diameter in a range of about 10 nm to about500 nm. The shell has a thickness in a range of about 10 nm to about 150nm. Additionally, each nanoparticle further comprises SERS reportermolecules hosted at the interface of the core and the shell, where theSERS reporter molecules comprises (1,2-bis(4-pyridyl)ethylene (BPE)).

Then, the suspension of the nanoparticles is carefully layered on thetop of the high-viscosity density gradient medium in the container.Subsequently, the layered suspension of the nanoparticles on the top ofthe high-viscosity density gradient medium in the container iscentrifuged at a predetermined speed for a predetermined period of timeto form a gradient of fractions/bands of the nanoparticles along thecontainer. Each fraction/band comprises nanoparticles in a respectiveone of aggregation states of the nanoparticles. Each fraction sortednanoparticles are collected from the container for use.

Accordingly the invention, the silica coating keeps the nanoantennasdispersed in aqueous solutions without functionalization or surfactants,which avoids complications in their use in potential technologicalapplications. The use of an aqueous iodixanol density gradient providesa sufficiently high solvent viscosity to compensate for their increasedmass, allowing sorting by aggregation state to occur. The particularlylow centrifugal forces required (about 500 g) and short times (about 10min.) underscore the potential scalability of this process.

In another aspect, the invention relates to a sensing platform thatutilizes the sorted nanoparticles as nanoantennas to sense. In oneembodiment, as shown in FIG. 7, the sensing platform 700 has a substrate710, an array of wells 720 formed in the substrate 710, and sortednanoparticles 730 filling in the array of wells 720. The sortednanoparticles are in a cluster state. The cluster state corresponds toone of a dimer state to a dodecamer state. In the exemplary embodiment,the majority of the sorted nanoparticles 730 filled in the array ofwells 720 is in a dimer state. Additionally, each well 720 contains asingle sorted nanoparticle 730. The sorted nanoparticles 730 arebiocompatible.

As disclosed above, each sorted nanoparticle 730 may have two or morecores and a shell encapsulating the two or more cores. Each of the twoor more cores is formed of a noble metal, such as gold. The shell isformed of a material such that the nanoparticles are dispersed in theaqueous solution without need for functionalization or surfactants. Inone embodiment, the shell is formed of silicon. Each sorted nanoparticlemay further comprises SERS reporter molecules hosted at the interface ofthe two or more cores and the shell, wherein the SERS reporter moleculescomprises (1,2-bis(4-pyridyl)ethylene (BPE)).

In a further aspect of the invention, a microfluidic device comprisesthe sensing platform disclosed above.

These and other aspects of the present invention are more specificallydescribed below.

Implementations and Examples of the Invention

Without intent to limit the scope of the invention, exemplary methodsand their related results according to the embodiments of the presentinvention are given below. Note that titles or subtitles may be used inthe examples for convenience of a reader, which in no way should limitthe scope of the invention. Moreover, certain theories are proposed anddisclosed herein; however, in no way they, whether they are right orwrong, should limit the scope of the invention so long as the inventionis practiced according to the invention without regard for anyparticular theory or scheme of action. It should be appreciated thatwhile these techniques are exemplary of preferred embodiments for thepractice of the invention, those of skill in the art, in light of thepresent disclosure, will recognize that numerous modifications can bemade without departing from the spirit and intended scope of theinvention.

Example 1 Sorting of Nanoparticles Via Centrifugal Processing

In this example, sample nanoparticles to be sorted and the sortednanoparticles were characterized by scanning TEM (STEM), UV-vis-NIRspectroscopy measurements and SERS ensemble measurements. Othercharacterization methods can also be used to practice the invention.

Electron microscopy characterization was performed on a Hitachi HD2300scanning transmission electron microscope (STEM), operating inhigh-resolution TE mode at 200 kV. Prior to observation, the sample wasprepared by drop-casting the solution of interest onto a TEM grid (300mesh formvar/carbon type B grid, Ted Pella, Inc.) and allowed to dry inair.

UV-vis-NIR spectroscopy measurements were carried out on a Cary 5000spectrophotometer (Varian, Inc.) operating in two-beam mode, where areference sample (aqueous iodixanol solution) was illuminated andmeasured concurrently with the sample, and the absorbance of thereference was subtracted from the sample measurement. A baselinecorrection was also used to account for variation in optical pathsbetween the beams. Spectra were obtained with a resolution of 1 nm andan integration time of 1.33 seconds.

SERS ensemble measurements were performed on 200-400 μL aqueoussuspensions of nanoantennas. All SER spectra were collected on acustom-built macro setup. The 632.8 nm excitation was obtained using aHeNe laser (12 mW output power, 6 mW at the sample) (ResearchElectro-Optics). The SERS measurements employ 1 in. interference andnotch filters (Semrock, Rochester, N.Y.), a single grating monochromatorwith the entrance slit set to 100 μm (Acton Research Corporation, Acton,Mass.), a liquid N₂ cooled charge-coupled device (CCD) detector (modelSpec10:400B, Roper Scientific, Trenton, N.J.), and a data acquisitionsystem (Photometrics, Tucson, Ariz.). The spectral positions of the CCDpixels were calibrated using cyclohexane.

In this example, the sample nanoparticles to be sorted areas-synthesized gold core/silica shell nanoparticles (Cabot SecurityMaterials, Inc.) having the SERS reporter molecules(1,2-bis(4-pyridyl)ethylene (BPE)) adsorbed at the gold/silica interfaceand exist in a variety of aggregation states.

A representative TEM image of the sample (HitachiHD2300 STEM operatingat 200 kV in TE mode) is shown in FIG. 1(a), where the nanoparticles arefound to exist as monomers or clusters that range from dimers tododecamers with approximately spherical gold cores (about 96±11 nm indiameter) encapsulated in a silica shell (about 63±4 nm). Monomersrepresent more than half (about 59%) of the sample population, as shownin the population histogram in FIG. 1(b). The minority species includedimers (about 17%), trimers (about 11%), tetramers (about 7%), andpentamers (about 3%), with the remaining clusters possessing more thanfive cores. The bulk extinction spectrum of the nanoantennas suspendedin water, as shown in FIG. 1(c) contains one peak centered at about 600nm and a broader band centered at about 950 nm. Monomers of the sizeused here have a single plasmon resonance band and contribute stronglyto the extinction at about 600 nm. Single particle LSPR (localizedsurface plasmon resonance) measurements have shown that dimers andtrimers have two plasmon resonance bands [1]. The shorter wavelengthband varies in position from about 650 nm to about 850 nm depending onevery structural detail, but is particularly sensitive to theinterparticle gap distance in the sub-2 nm range. Similarly, the widthof the resonance depends sensitively on the structural details. Thelonger wavelength band varies in position from about 800 nm to about1000 nm and is also structure sensitive. Consequently, dimer, trimers,and probably tetramers contribute to the extinction in the tail of theensemble band at about 600 nm as well as to the blue leading edge of theabout 950 nm band. Multi-core clusters contribute the rest of theextinction for the about 950 nm band. While the multi-core clusters havedemonstrated excellent SERS enhancement in previous correlatedstructure-SERS activity measurements performed on similar nanoparticlesdiffering only by their reporter molecule [1], no surface-enhanced Raman(SER) signal is measured from monomers at low excitation power.

In this example, aqueous density gradients were formed using Optiprep60% w/v iodixanol, 1.32 g cm⁻³ (Axis-Shield, PLC). Gradients were formedin centrifuge tubes using a linear gradient maker (SG 15 Linear GradientMaker, Hoefer, Inc.) with about 5 mL starting solutions of about 30% andabout 60% w/v iodixanol. About 200 μL of bath sonicated goldnanoparticle solution (0% iodixanol) was then carefully layered on thetop of the gradient using a syringe and 23 gauge needle.

Centrifugal sorting of the as-synthesized gold core/silica shellnanoparticles was accomplished using a Beckman SW41Ti swinging bucketrotor. The centrifuge tubes were initially loaded with a linear densitygradient of about 30%-60% weight per volume iodixanol (about 1.16-1.32 gcm⁻³). Then, about 200 μL of an aqueous suspension of the as-synthesizedgold core/silica shell nanoparticles were carefully layered on the top.Importantly, the silica coating allows the nanoparticles to be welldispersed in water without the need for additional chemicalfunctionalization or surfactants. After centrifuging at a relatively lowspeed (with a low centrifugal force of about 500 g) for about 10minutes, a well-defined band and subsequent gradient of material isobserved, as shown in FIG. 2(a).

Millimeter fractions were collected from the tube for TEMcharacterization using a piston gradient fractionator (BiocompInstruments, Inc., Canada), and the resulting extinction spectra andaggregation state histograms based on particle counting from the TEMimages for three of the fractions, labeled (i), (ii), and (iii), areshown in FIG. 2. The extinction spectra were measured by UV-vis-NIRspectroscopy in solution for the collected fractions and are shown inFIG. 2(b). In the top band (i), only the surface plasmon band near 570nm is present. On the other hand, for fractions at lower positions inthe centrifuge tube, a new broad band in the near-IR known to correspondto multi-core aggregates [33] is apparent, and the surface plasmon bandis broadened and red-shifted. These spectra thus provide evidence thatsorting by nanoparticle aggregation state has occurred with the top bandlikely enriched in monomers and subsequent bands possessing nanoparticleclusters. Analysis of the TEM images, as shown in FIG. 2(c), and itshistograms, as shown in FIG. 2(d), verifies this assignment by directlyshowing that the top band (i) contains almost exclusively monomers(about 97%), while subsequent fractions possess an increasing quantityand size of nanoparticle clusters. In particular, fraction (ii) includesdimers (about 52%), trimers (about 32%), and monomers (about 13%), whilefraction (iii) includes tetramers (about 32%), pentamers (about 28%),trimers (about 21%), dimers (about 9%), and monomers (about 3%). Eventhough monomers are found outside the top band, at a sufficient tubedepth, they are reduced to a negligible proportion of the population,thus implying fractions that consist almost exclusively of SERS-activenanoantennas.

While the sorting method is highly effective at both removing monomersand targeting a narrow range of aggregation states, fractions other thanthe top band contain more than one nanoparticle aggregation state. Todetermine if this remaining polydispersity resulted from insufficientspatial separation of the bands in the centrifuge tube and/or imprecisefractionation, the centrifugation time was increased to spread thesorted material over a larger length of the centrifuge tube. Sortingexperiments were carried out using the same sample and gradientpreparation as described above for the original separation.Centrifugation time was doubled from about 10 minutes to about 20minutes to allow bands of sorted material to spread further down thecentrifuge tube, as shown in FIG. 3, where centrifuge tube images (a)and (d) of separations are for the centrifugation times of about 10minutes and about 20 minutes, respectively. Corresponding fractions arespatially indicated, each accompanied by a sample TEM image (b)/(e) andhistogram (c)/(f) of aggregation states. A modest improvement in sortingis observed, although the concentration of dimers remains under 75%.

Since the experiment provided minimal refinement compared to theoriginal separation, the nanoclusters of differing aggregation stateslikely possess overlapping sedimentation coefficients. For example, thevariability in the gold core diameter and shell thickness yields adistribution of nanoparticle cluster mass and volume. The effect of thestructural polydispersity on the sedimentation coefficient distributionis exacerbated at higher nanoparticle aggregation states where multiplegold cores compound the variability in the nanoparticle cluster mass andvolume. Furthermore, nanoparticle clusters including multiple gold corescan adopt a range of geometries (i.e., aspect ratios), which alsoinfluences the sedimentation coefficient.

To quantify the polydispersity in sedimentation coefficient as afunction of nanoparticle aggregation state, a large number ofnanoparticles (greater than 600) from the as-synthesized sample wereimaged in TEM. The core diameters and aspect ratios were thenindividually extracted from the TEM data. The sedimentation coefficientcan then be calculated using the following equation:

${s = \frac{m\left( {1 - {\rho_{s}/\rho_{p}}} \right)}{f}},$where m is the total particle mass, ρ_(s) and ρ_(p) are the densities ofthe solvent and particle respectively, and f is the frictionalcoefficient, which depends on the shape of the nanoparticle and theviscosity of the solvent. Aggregates larger than dimers possess avariety of shapes, requiring a thorough analysis to account for varyingaspect ratios. To account for the observed variation in shape, as shownin FIG. 4, the nanoparticles were modeled as prolate ellipsoids usingthe total volume of gold obtained from the core diameters, the averagesilica shell thickness, and the individually measured aspect ratios. Inone embodiment, the aspect ratios were measured for each individualaggregate and included in the final sedimentation coefficient analysis.

The frictional coefficient was then calculated using the Perrin equationfor frictional ratios of ellipsoids of revolution [34], which modifiesthe frictional coefficient of a sphere with a geometrical correctionfactor P as follows:f _(ellipsoid) =P(3πηd),

where P=(1−q²)^(1/2)/└q^(2/3)(ln({1+(1−q²)^(1/2)}/q))┘ Here, η is theviscosity of the solvent, d is the effective diameter of the equivalentsphere, and q is the aspect ratio of the prolate ellipsoid where q<1.

Using this ellipsoidal model and the structural parameters extractedfrom TEM, the distribution of sedimentation coefficients was calculatedfor the as-synthesized sample at a fixed point (about 40% w/v iodixanol,about 1.21 g cm⁻³) in the density gradient [35], as shown in FIG. 5.This model reveals the presence of a monomer band at low sedimentationcoefficients, followed by a gap in sedimentation coefficient, andfinally overlapping aggregation states at higher sedimentationcoefficients. Consequently, transient centrifugal sorting is expected toyield highly enriched monomers at the top of the centrifuge tube andthen increasing but overlapping levels of nanoparticle aggregation forsubsequent fractions, in agreement with the experimental results. Thismodel thus holds promise for evaluating the feasibility and/or refiningthe experimental conditions for sorting other nanoparticle clusters viatransient density gradient centrifugation techniques, assuming that theinitial nanoparticle structural parameters and polydispersity have beendetermined. On the other hand, the results of transient density gradientcentrifugation experiments can provide quantitative insight into thestructural polydispersity of nanoparticle samples.

To show the utility of the sorted nanoparticle clusters, ensembleaveraged SERS measurements were performed on the initial solution andthe selected fractions (i), (ii), and (iii). The nanoparticleconcentrations were balanced between the samples using the visiblesurface plasmon band in the extinction spectra. The SER spectra of1,2-bis(4-pyridyl)ethylene (BPE) were collected using excitation at awavelength of about 632.8 nm and power of about 6 mW (HeNe laser 17 mWfrom Research Electro-optics). The spectra were acquired for about 15seconds with 3 accumulations. The fraction enriched in monomers (i)exhibited no peaks, i.e. no SER signal was observed (spectrum (a), FIG.6), which is in agreement with results published earlier on similar SERSnanoantennas [1]. The initial solution (spectrum (b), FIG. 6), exhibitedthe characteristic peaks of BPE including 1616 cm⁻¹, 1643 cm⁻¹, 1203cm⁻¹, 1341 cm⁻¹, and 1024 cm⁻¹ in decreasing intensity. The same peakswere observed with the same intensity ratio on the measured SER spectrafrom the fractions enriched in dimers and trimers (ii) (spectrum (c),FIG. 6) and tetramers (iii) (spectrum (d), FIG. 6). The intensities ofthe peaks from the sorted fractions, where the amount of monomers hasbeen substantially reduced, are higher than for the initial solution.This increase in SER signal is further evidence that centrifugal sortinghas yielded samples enriched in SERS-active nanoantennas.

Both the nanoparticle cluster sorting technique and the resultingrefined populations of plasmonic nanoantennas can find a wide range ofpotential applications. This sorting mechanism can be applied to a vastarray of nanoscale core/shell structures, allowing for improvedpopulation control over particles too massive for current centrifugalsorting techniques. There are currently numerous applications fornanoparticles, including drug delivery, industrial coatings, batteryanodes, and solar cells, which could benefit from greater control overnanoparticle population and enable larger structures to be used withoutnecessitating large variation in particle size and shape. Core/shellnanoparticles specifically have found applications in optical sensing,MRI, fluorescence imaging, pigments, and catalysis. Plasmonicnanoantennas in particular are currently being used in the securityindustry for unique tagging and sensing applications, and moremonodisperse samples could yield higher detection signals and improvedreliability and reproducibility between samples.

Example 2 Sensing Platform

In this exemplary embodiment, a sensing platform that utilizes thecentrifugally sorted core/shell nanoantennas for detecting andeliminating blood-borne pathogens is shown. This work is focused towardcreating a dialysis-like therapeutic system whereby blood cycles througha portable device, is continuously sensed, and pathogens are removed viamanipulation and separation of the fluid. The sorted core/shellnanoantennas have demonstrated a dramatic improvement in sensingcapabilities by amplifying a key parameter, a percentage of activesensing sites, and are now being incorporated into the design of thissystem.

The sensing component of the sensing platform device includes asubstrate containing an array of wells, into which the nanoantennas areinserted via fluid deposition. The surface design and deposition havebeen optimized such that nearly every well is filled with a nanoantenna.

The sensing capabilities of the substrate come from the nanoantennasthemselves, whose nature and surface coverage are critical forhigh-performance sensing. After the nanoantennas are deposited fromsolution onto the substrate, their performance is assessed by SERS andthe contents of the wells are quantitatively evaluated via SEM. Bysorting the initial nanoantenna population to maximize the depositednumber of active nanoantennas (dimers, trimers, . . . ) and minimize thenumber of inactive species (monomers), the sensing capabilities of theresulting welled substrate can be increased.

Referring to FIG. 7, a sensing platform 700 is schematically shownaccording to one embodiment of the present invention. The sensingplatform 700 includes a substrate 710 having a top surface 712 and anopposite, bottom surface 714, an array of wells 720 formed on the topsurface 712 of the substrate 710, and sorted nanoantennas 730 filling inthe array of wells 720. The sorted nanoantennas 730 can be in a clusterstate as dimers, trimers, . . . , or dodecamers. In the exemplaryembodiment shown in FIG. 7, the sorted nanoantennas 730 are in a dimerstate.

After depositing centrifugally sorted core/shell nanoantennas 730 ontothe wells 720 on the top surface 712 of the substrate 710, it wasobserved that the population of wells containing inactive nanoantennasdecreased from about 72% to about 11% as compared to unsortedas-synthesized nanoparticles, and the population in the wells 720containing high-performance dimer and trimer nanoantennas increased fromabout 26% to about 86%. This dramatic improvement is shown in FIGS. 8and 9.

FIG. 8 shows an SEM image (a) of the array of wells containing unsortednanoantennas of the sensing platform, and its corresponding histogram ofthe pollutions (b), where the vast majority of the populations in thearray of wells is monomers, i.e., inactive.

FIG. 9 shows an SEM image (a) of the array of wells containing thecentrifugally sorted nanoantennas, and its corresponding histogram ofthe pollutions (b), where the vast majority of the populations in thearray of wells is dimers, trimers, . . . , i.e., active, while thepopulations of inactive species (monomers) are minimal.

After optimizing the sensing capabilities of the sensing platform usingthe sorted nanoantennas, this architecture has been successfullyintegrated into a microfluidic device and SERS spectra have beenobtained. This demonstrates that the nanoantenna-in-wells geometrycombined with using the core/shell sorting method is both compatiblewith and enhances the capabilities of the microfluidic device.

FIG. 10 shows an optical microscope image (a) of such a microfluidicdevice incorporating a nanoantenna-in-wells substrate (inset). Ramanspectra (b) have successfully been measured, demonstrating that thisdetection system is compatible with the microfluidic architecture.

In summary, the present invention, among other things, recites a facile,surfactant-free method for sorting high-mass silica-coated goldnanoparticle clusters by aggregation state via transient densitygradient centrifugation. The improved monodispersity of the nanoparticleclusters is quantified by TEM, extinction spectroscopy, and SERS.Furthermore, a quantitative model is presented that accurately mirrorsthe observed sorting results and provides guidance to future efforts toseparate other nanoparticles in the transient sedimentation regime. Byremoving non-SERS-active monomers and narrowing the nanoparticleaggregation state distribution, density gradient centrifugation servesas an effective post-synthetic processing technique for realizinguniform and reproducible structures for plasmonic nanoantennaapplications such as SERS-based sensors.

Both the nanoparticle cluster sorting technique and the resultingrefined populations of plasmonic nanoantennas can find a wide range ofpotential applications. This sorting mechanism can be applied to a vastarray of nanoscale core/shell structures, allowing for improvedpopulation control over particles too massive for current centrifugalsorting techniques. The sorted nanoantennas can be utilized in sensingplatform and microfluidic devices that are particularly effective attreating sepsis, a life-threatening condition where the bloodstream isoverwhelmed with bacteria, wound infection, autoimmune diseases, cancer,diabetes, and regenerative medicine.

The foregoing description of the exemplary embodiments of the inventionhas been presented only for the purposes of illustration and descriptionand is not intended to be exhaustive or to limit the invention to theprecise forms disclosed. Many modifications and variations are possiblein light of the above teaching.

The embodiments were chosen and described in order to explain theprinciples of the invention and their practical application so as toenable others skilled in the art to utilize the invention and variousembodiments and with various modifications as are suited to theparticular use contemplated. Alternative embodiments will becomeapparent to those skilled in the art to which the present inventionpertains without departing from its spirit and scope. Accordingly, thescope of the present invention is defined by the appended claims ratherthan the foregoing description and the exemplary embodiments describedtherein.

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What is claimed is:
 1. A sensing platform, comprising: (a) a substrate;(b) an array of wells formed in the substrate; and (c) sortednanoparticles filling in the array of wells such that each well containsa single sorted nanoparticle, wherein the sorted nanoparticles are in acluster state, wherein each sorted nanoparticle comprises two or morecores and a shell encapsulating the two or more cores.
 2. The sensingplatform of claim 1, wherein each of the two or more cores is formed ofa noble metal.
 3. The sensing platform of claim 2, wherein each of thetwo or more cores is formed of gold.
 4. The sensing platform of claim 1,wherein the shell is formed of a material such that the nanoparticlesare dispersed in the aqueous solution without need for functionalizationor surfactants.
 5. The sensing platform of claim 4, wherein the shell isformed of silica.
 6. The sensing platform of claim 1, wherein eachsorted nanoparticle further comprises surface-enhanced Raman scattering(SERS) reporter molecules hosted at the interface of the two or morecores and the shell.
 7. The sensing platform of claim 6, wherein theSERS reporter molecules comprises (1,2-bis(4-pyridyl)ethylene (BPE)). 8.The sensing platform of claim 1, wherein the sorted nanoparticles arebiocompatible.
 9. The sensing platform of claim 1, wherein the clusterstate corresponds to one of a dimer state to a dodecamer state.
 10. Amicrofluidic device comprising the sensing platform of claim 1.